Francisella tularensis is a highly infectious bacterium responsible for tularemia, a disease whose pneumonic form has potentially lethal consequences in humans. Francisella virulence depends on its ability to survive and replicate inside macrophages of the infected host. The current model of Francisella intracellular fate is initial enclosure within a phagosome, followed by escape from this phagosome and then replication in the cytoplasm, but the bacterial determinants controlling these individual stages are unknown. A Francisella pathogenicity island (FPI) has been identified as required for intracellular growth but how it contributes to Francisella virulence is not understood. We have been using cell biology-, bacterial genetics- and genomics-based approaches to further characterize Francisella intracellular trafficking, identify genes expressed at various stages of the intracellular cycle and assess their role in Francisella virulence. In a model of murine primary macrophage infection with the F. tularensis type B attenuated LVS strain, we have shown that phagosomal escape occurs rapidly after phagocytosis (~ 20 min) using a fluorescence microscopy-based assay to measure phagosome integrity. Unexpectedly, we have found that bacterial replication in the cytoplasm is followed by the enclosure of bacteria within a membrane-bound compartment (~ 24 h) that displays properties of mature autophagosomes, indicating that autophagy is involved in Francisella intracellular trafficking. Furthermore, formation of these late Francisella-containing vacuoles (FCV) requires bacterial replication and protein synthesis, suggesting that this process is controlled by the replicating bacteria. Type A virulent clinical isolates of F. tularensis showed a similar, although faster, intracellular trafficking in murine macrophages and also formed late FCVs, suggesting a common mechanism between type A and B strains. To examine whether autophagy is necessary to Francisella intracellular survival and determine the role of late FCVs in Francisella virulence, we are currently using small inhibitory RNA technology to specifically knock down proteins controlling autophagy and assess the effect on Francisella intracellular behaviour. In collaboration with Francis Nano (University of Victoria), we have also investigated the role of individual genes of the FPI in intracellular trafficking, using deletion mutants of F. novicida. Our results indicate differential roles of FPI genes in various stages of Francisella intracellular cycle, such as phagosomal escape (igl genes) and cytoplasmic replication (pdp genes). Future work aims at pursuing the characterization of the phenotypes associated with mutations of igl and pdp genes to further define their intracellular roles in light of our expanding knowledge of the intracellular Francisella lifecycle.